Spectroscopy is used in a variety of fields to determine the compositions of solutions by identifying and measuring the wavelengths and intensity of electromagnetic radiation absorbed or emitted by compounds in the solution. Spectroscopy is particularly useful for identifying, characterizing and quantifying compounds that absorb in the ultraviolet (U.V) or visible portion of the spectrum because many biologically important macromolecules, including biopolymers, such as DNA, RNA, proteins and carbohydrates, absorb energy in at least one region of the spectrum of U.V. or visible portion of the electromagnetic spectrum. Under appropriate conditions it is possible, using spectrometry to detect, identify and measure the concentration of such compounds in solution.
Upon exposure of a solution to incident light that includes wavelengths that have energy that matches the difference between two allowed states of compounds in the solution, photons of the particular wavelength are absorbed so that the component wavelengths of the transmitted light differ from those of the incident light. Following absorption, the electrons in the compounds revert ground state by loss of energy to collisions and other heat generating interactions. For some compounds, however, particularly those that have conjugated electrons, reversion to ground state is slower and includes the emission of photons. Depending upon the nature of the excited state the emission may result in fluorescence. Fluorescent emissions have a lower energy level than the incident absorbed light and may be detected with high sensitivity by a photodetector, as long the incident light does not interfere with the emitted light. Typically, in fluorescence spectrometers the photodetector is placed at an angle, usually at right angles, to the incident light. When absorption or emission is in the U.V and visible region of the spectrum, such transitions are particularly suitable for measurement by photometric methods, including absorbance spectrophotometry and fluorescence spectrophotometry. In practice, a sample of a solution containing a compound, such as a biopolymer, is introduced into a cuvette, a container which is transparent to the wavelength absorbed by the compound of interest, and the cuvette is placed into a spectrometer, such as a spectrophotometer or spectrofluorimeter. Absorbance spectrophotometry and fluorescence spectrometry (spectrofluorimetry) involve the measurement of electronic transitions of compounds, either by absorption or emission or electromagnetic radiation, and permit the detection, characterization and quantification of such compounds in solutions. The energy at which absorption occurs is a function of the electron, vibrational and rotational energy levels of the compounds in the solution.
In order to enhance or alter the light absorption or light emission characteristics of a particular compound or to detect a particular constituent of a solution or mixture of compounds, it is sometimes necessary to add an appropriate reagent, which specifically interacts with the chemical or compound of interest, to the solution prior to analysis. The reagent specifically interacts with the chemical or compound of interest to form a complex or other product that has different absorption or emission properties than either the compound or reagent alone. The reagent is selected to combine with a particular compound or precursor of the compound that is present in the solution to form a complex that has enhanced light absorption or light emission characteristics compared to the compound in the absence of the reagent and that can be detected using an absorbance spectrophotometer, fluorescence spectrometer, or other photometric instrument, or even observed with the naked eye. For example, highly fluorescent compounds are relatively rare and may be used as reagents for tagging non-fluorescent or weakly fluorescent compounds. A fluorescent compound can be bound or otherwise complexed with a non-fluorescent compounds and thereby provide a means for detecting, identifying and quantifying such non-fluorescent compounds.
Absorbance is directly proportional to concentration so that the concentration of particular compounds may be determined from absorbance measurements. Fluorescence is directly proportional to concentration only in dilute solutions. It is generally a function of several variables, including incident intensity and the particular instrument used. Concentrations of fluorescent compounds may be measured using fluorescent spectrometry by preparing a standard curve.
In particular, in studying samples of body fluids or solutions of biopolymers that contain compounds, such as DNA, RNA, proteins and carbohydrates, detection and quantification of the concentration of the compound is improved by first contacting the solution with a reagent such as a dye or fluorescent compound, that reacts with a particular compound in solution and alters or enhances the absorbance or emission of electromagnetic radiation by the compound.
In order to conduct typical spectrophotometric and spectrofluorimetric and other photometric assays for macromolecules, a specific reagent is added to a sample solution that contains the compound of interest. The reagent interacts with the macromolecule to form a complex that is directly or indirectly detectable a characteristic absorbance or fluorescence profile. The reagent forms a complex or other product that absorbs, emits or produces electromagnetic radiation of a specific or particular wavelength that can be used to detect, identify or quantify the concentration of the compound of interest in the sample solution. Such reagents include, dyes, such as Hoechst dye, (2-(2-(4-hydroxyphenol)-6-benzymidazoyl-6(1-methyl-4-piperazyl)benzimidazo le), which interacts with DNA to form a complex that has increased fluorescence, and various dyes, such as Coomassie blue, that bind to proteins and result in colored products that absorb strongly in the visible portion of the spectrum. The concentration of the macromolecule, such as DNA or protein, may be determined by measuring the absorbance or emission at selected absorbance or fluorescence maxima.
Since detection of the particular compound requires modification of the compound, the sample may be discarded after the measurement is made. Since the amount of sample that is available for study is often very limited, it is desirable to use as little of the sample as possible for spectroscopic analysis. The amount of sample that is used, however, is constrained by the limits of detection of the system. Generally, as the size of the cuvette is decreased, the amount of sample is reduced, which in turn decreases the size of the signal produced upon exposure of the sample to light emitted by the spectrophotometer, thereby decreasing the sensitivity and accuracy of the measurement. If the solution is introduced into a standard cuvette, which is about 12.5 mm.times.12.5 mm and which must be completely filled for accurate measurements, and if the limits of detection are in the 1-10 .mu.g/ml range, then a substantial portion of the available sample may be depleted by spectrometric analysis.
U.S. Pat. No. 4,991,958 and U.S. patent application Ser. No. 07/433,752 to Garner, which have herein been incorporated in their entirety by reference thereto, describe adaptors for spectrometric analysis that are designed to properly focus light onto a sample held in a micropipette that is mounted in the adaptor in a spectrophotometer and in a fluorescence spectrometer, respectively. By virtue of the design, the adaptors provide means for spectrometric measurements of small samples. Each adaptor is designed to focus light from the spectrometer light source along the axis of the micropipette and to refocus light emitted from or through the pipette so that it is received by the detector. U.S. Pat. No. 4,991,958 and U.S. patent application No. 07/433,752 to Garner, however, provide little guidance regarding the optimal design of micropipettes for use in the adaptors.
In addition, since many spectrometric assays for macromolecules rely on the addition of regents to render the macromolecule detectable, the reagents must be premixed with the sample solution to form detectable products. Such pre-mixing of reagent and the sample creates other technical complications, including exposing the analysts to physically harmful chemicals, such as ethidium bromide. Also, typically the reagents are added and mixed in cuvettes. Because cuvettes are quartz or glass or other suitable material that is transparent to a particular range of wavelengths, and, because they are optical components of the spectrometer, they must be engineered to fairly exacting specifications. Consequently cuvettes, generally, are not disposable. Such reusable cuvettes or other containers may become contaminated and hence unsuitable for subsequent use. Additionally, such reuse can result in contamination of the sample.
Thus, there a need to provide a receptacle that can hold a relatively small sample for analysis within a spectrometer. In addition, in order to facilitate spectroscopic analysis, there is a need to provide a means by which a reagent can easily and safely be mixed with the relatively small amount of sample that is held within the micropipette and to provide means for accurately detecting and quantifying the concentrations of particular compounds that are present in samples in relatively low concentrations and in relatively small volumes.
Therefore, it is an object of the present invention to provide a micropipette that is designed to hold a relatively small sample of the liquid in a spectrometer.
It is a further object of the present invention to provide a micropipette mountable in a spectrophotometer that holds a liquid sample in the spectrometer and that includes means for safely and easily mixing a reagent with the sample solution.
Another object of the present invention is to provide a micropipette for holding a liquid sample in a spectrometer in which a reagent can be pre-mixed with a compound in solution in a reproducible and standardized procedure.
It is another object is to provide a disposable micropipette for holding a sample solution in a spectrometer. A further object is to provide a micropipette for holding a liquid sample in a spectrometer which reduces the likelihood of contamination of the liquid held in the micropipette. Another object is to provide a micropipette, for holding a sample solution in a spectrometer, that is relatively easy to use and comparatively cost-effective to manufacture.